FerTech Lect 8 Alcohol

Tekfer14_Lecture8 9/3/2015
ITP 321 Lecture Note 8
Alcohol Fermentation
Lilis Nuraida
Department of Food Science and Technology
Bogor Agricultural University
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Alcohol fermentation
• Alcohol fermentation is an anaerobic
(no oxygen), exothermic (heat-
releasing) process in which sugars
break down into ethyl alcohol
CH3CH2OH and carbon dioxide gas
C6H12O6 -->
> 2 C2H5OH + 2 CO2 + 23 Kcal
In yeast, when O2 is available, cells use aerobic cellular

respiration to transfer energy from the
organic molecules to ATP.
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Reversible reactions
Acetaldehyde is an important component in many

industrial fermentations, particularly for food and
alcohol
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Alcoholic fermentation
• When yeast is grown
under anaerobic
conditions, sugars are
fermented to alcohol
and carbon dioxide.
• At about a
concentration of 12%,
ethanol itself inhibits
further growth of the
microbial organisms
and the process stops.
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Key microorganism
of alcoholic
fermentation is
Saccharomyces
Saccharomyces,
unable to
breakdown starch
 need help other
microorganism
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Saccharomyces yeasts
• Mankind’s oldest domesticated microorganism
• Considered to be the best studied and most
Considered to be the best studied and most
used yeast:
– 30 million tons of wine p.a.
– 60 million tons of beer p.a.
– 600,000 tons of baker yeast p.a.
– 800,000 tons of single cell protein p.a.
Verstrepen et al. 2006
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Technological Function of Yeast
• Dough Leavening (baker’s yeast)
• Brewing (brewer’s yeast)
• Distilled alcohol beverages fermentation (distiller’s
yeast)
• Wine fermentation (wine yeast)
• Other alcoholic fermentation (ragi)
– Indonesia: tape, brem
d i b
• Dairy fermentation (kefir grain)
• Soy fermentation (soy sauce, tauco)
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Factor affecting yeast activity
• Availability of major and minor nutrient
• Temperature
– S. cerevisae optimum growth at 30 C
• pH
• Dissolve oxygen:
– When oxygen presence, glucose is metabolised by
aerobic respiration
aerobic respiration
• Possible presence of competing microorganism:
– Apply good sanitation
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Fermentation of alcoholic drink and
food
• Traditional Indonesia:
– Tape
– Brem
– Tuak
• Asia:
– Rice wine
– Coconut wine
• Europe, USA:
– Wine
i
– Beer
– Etc.
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Traditional fermentation‐1
• Brem
– Made of liquid of fermented glutinuous rice
• Filtered and bottled
– Brem cake
• The liquid is evaporated
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• Use of starter culture (ragi)
– Consist of consortium of microbes incl. mold,
yeast and lactic acid bacteria
– Starch is converted to sugars prior to alcohol
fermentation
– Fermentation is under anaerobic condition
• Sealed container
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• Tape fermentation
– Cooked cassava or cooked glutinuous rice
– Innoculated with starter
– Keep in close container
– Allow to ferment for 4‐6 days
– Prolong fermentation will increase alcohol
Prolong fermentation will increase alcohol
concentration
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Preparation of tape starter culture: home scale

Rice flour
Spices : pepper, chilli,
cinamon, garlic and
galangal powder
Roasting
Cooling
Water, coconut water or
sugar cane water
Mixing
Inoculation with commercial tape starter
Molding : round
Ragi/Tape
(cookie)-shaped, Drying
starter
incubation for 48
hours
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Microbiology of Ragi
• Predominant species :
– Yeast:
Yeast: Wickerhamomyces
Wickerhamomyces anomalus (Candida
(Candida
pelliculosa), Saccharomycopsis fibuligera
• Saccharomycopsis fibuligera ferment glucose, maltose and
sucrose and are able to assimilate glucose, maltose, sucrose,
cellobiose and soluble starch
– Molds, Amylomyces rouxii,
• Amylomyces strains utilise glycerol, maltose, sucrose and
g
soluble starch, and form lactic acid from glucose
– LAB: Pediococcus pentosaceus and Weisella spp. were
the predominant lactic acid bacteria in ragi while
Lactobacillus curvatus and Enterococcus faecium were
associated with certain types of ragi.
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Microbiology of Tape
• The important microorganisms for tape ketan fermentation are
Amylomyces rouxii and at least one species of yeast.
• The yeasts most commonly found in tape are
– Hyphopichia burtonii (Endomycopsis burtonii),
– Saccharomycopsis fibuligera (Endomycopsis fibuligera) and
– Candida beverwijkiae (Candida pellicullosa).
• Other yeasts that may also be present include:
– Saccharomyces cerevisiae
– Wickerhamomyces anomalus (Hansenula anomala),
– Candida beverwijkiae (Candida pellicullosa),
– Wickerhamomyces anomalus (Hansenula anomala).
• Lactic acid bacteria:
– Weisella spp. growing first, followed by Pediococcus pentosaceus.
– Enterococcus spp. developed concurrently with Weisella and Pediococcus.
Lactobacillus spp. found after 24 h
– Acetobacter is also found
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Tape fermentation
• Mixed fermentation of mold, yeast and lactic acid bacteria
• Mold incl. Mucor, Amylomyces rouxii etc.
– Convert starch into sugars
• Amylomyces rouxii are strongly amylolytic organisms
• Yeast, Saccharomycopsis fibuligera, Sacch. cerevisiae:
– Convert sugars into alcohol
• Saccharomyces cerevisiae are not amylolytic and, hence, their growth
in starchy materials depends on other microbes generating sugars and
other nutrient.
• S. cerevisiae
S. cerevisiae is a strong sugar fermenter
is a strong sugar fermenter and, being ethanol tolerant,
and, being ethanol tolerant,
produces high ethanol concentrations
• Candida beverwijkiae are strongly amylolytic organisms
• Lactic acid bacteria:
– Convert sugars into lactic acid
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Taxonomy of Alcoholic Beverages
• Wines – produced by fermentation of
fruit juices.
• Spirits – produced by distillation of a
wine or beer.
• Beers –
B produced by fermentation of
d db f t ti f
grains or starchy staples.
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Specialty Wines
• Champagne and other sparkling wines have
carbon dioxide in bottle.
b di id i b ttl
• Aromatic wines include plant extracts such as
coriander, bitter orange, wormwood.
• Fortified wines have higher alcohol content
(15‐21%) because brandy or other distilled
(15‐21%) because brandy or other distilled
alcohol is added.
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Wine Yeast
• Controlled fermentation started in 20th century
y
• Natural fermentation involves different yeast
species such grow succesively at initial.
– In the most active fermentation stage to end
of fermentation: Saccharomyces cerevisiae
– Other species: S. bayanus
• Saccharomyces
y converts the glucose,
g ,
fructose and sucrose found in grape must
and juice into ethanol
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Desirable characteristics of wine Yeast
• Reasonable rate of fermentation
• Predictable fermentation characteristics
• Good ethanol tolerance
• Good temperature tolerance
• Sulfur dioxide tolerance
• Little to no off‐character production
– Hydrogen sulfide
– Acetic acid
– Ethyl carbamate
• Little to no inhibition of other desirable microbes
• Killer factor resistant
• Production of desired aroma characters
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Desirable properties of Distiller’s Yeast
• Natural
Natural fermentation: any number of
fermentation: any number of
species may be involved
• Controlled fermentation: S.cerevisiae
– Very alcoholic tolerant and gives a good yield
– Ferments rapidly
– Good flavor production
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Yeast Preparation based on Activity
• Yeasts are available in several forms, and
are classified on the basis of their activity.
l ifi d th b i f th i ti it
– Active yeasts include:
• Baker’s yeast
• Brewer’s yeast
• Yeasts for alcoholic beverages
– Inactive yeasts, such as:
• Dried brewer’s yeast
• Primary‐ grown yeasts
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Yeast Starter Culture Preparation
• Yeast Cream (18‐20% solid) Perishable, kept
• Compressed yeast (27‐32% solid) refrigerated
• Dry yeast (8% moisture) Shelf-life up to 2 y
– Active Dry Yeast in suitable
– Instant Dry Yeast packaging
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Storage Stability of Active Dry Baker’s Yeast in
Variuos Atmosphere
Atmosphere Residual Activity (%)*)
Nitrogen 79.5
Carbon dioxide 81.9
Carbon monoxide 85.2
Argon 80.7
Hydrogen 88.9
Air 43 3
43.3
Vacuum 70.3
*) after storage for 3 d at 55oC
Hill, 1987
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Technologically Important Properties‐1
• Ethanol tolerance
– Saccharomyces is the most tolerance
is the most tolerance
• How Does Ethanol Inhibit Yeast?
– Displaces water of hydration changing the properties
of protein‐lipid interactions
– Denatures proteins
– Disrupts protein active sites
– Damage to the cell membrane
Damage to the cell membrane
• Allows increased passage of protons from the medium into
the cell leading to acidification of the cytoplasm
• Removal of protons requires expenditure of energy
– End product inhibition of glycolytic enzym
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• Floculation
– Permits ready separation of the yeast from
y p y
the product at the end of the fermentation
• Minimizes off flavour
– To rapid floculation can result in failure
fermentation
– Floculation involve formation of open
agglomeration of cells, occurs in absence of
cells division and involves divalent ions
cells division and involves divalent ions
(Ca2+), forming anionic groups at the cell
surface
• Difference flocculation characteristics
result from difference cell wall structure
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• Reistance to killer activity
– A number of strains of Saccharomyces
A number of strains of Saccharomyces
produce extra‐cellular toxin, zymocin
• Lethal to other strain (killer‐sensitive
strains)
– Killer activity is beneficial in suppressing wild
yeasts
• Attempt has been made by introducing the
properties using genetic engineering
technique
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Wines
• Wine grape is usual source of juice.
– native to Eurasia
– glucose and fructose are the usual fruit sugars
• Fermentation simpler than beer
Fermentation simpler than beer
• Other wines named for fruit used, such as cherry
wine or apple wine.
• Wines generally 9‐14% alcohol.
• Yeast killed by alcohol levels above 15%.
• Type:
– Sweet or dry depending on level of sugar left in
Sweet or dry depending on level of sugar left in
wine after fermentation
• Dry (0.2% RS)
• Semisweet (0.5‐2% RS)
• Sweet (5‐10% RS)
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Alcohol fermentation of wine
• During wine fermentation:
– Phenolic compound present in the grape may
Phenolic compound present in the grape may
interfere respiration. Ethanol is also inhibitory to
respiration
– Efficiency of ATP yield is only an issue if sugar is
limiting factor
• High
High sugar concentrations repress the
s gar concentrations repress the
synthesis of mRNA from genes that are
involved in respiration and oxidative
metabolism
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The Basic Steps of Red Wine

Production
1. Crushing, destemming
2. Must to tank
Optimal T:
3. Fermentation/Maceration 24-27 C
3-5
3 5d
4. Pressing and Draining Excessive temp
inhibits yeast and
5. Finish of Fermentation permits other
microorganisme to
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The Basic Steps of White Wine

Production
1 Crushing,
1. Crushing destemming
2. Pressing
3. Cold Settling/Racking Optimal T:
10-21 C
4 Fermentation
4. 7-14
7 14 d
5. Racking
6. Finish of Fermentation
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Factors affecting wine fermentation
• Temperature 18 C ‐ 24 C
• Sugar (but not more than about 25%)
g ( )
• Acidity: pH 3.5 ‐ 5.5
• Nutrients (source of N, vitamins: biotin, Mineral and
Trace Elements: Mg, Ca, Mn, K, Zn, Fe, Cu)
• Tannin (appropriat conc.)  fungal inhibitor
• Low oxygen (The first part of fermentation needs oxygen
for yeast replication)
• I
Innoculation Practices
l i P i
• Winery Practices
Grape is just a right substrat

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• Grape Berry Microflora
– 95‐98% of total organisms are molds and bacteria
– 2‐5% are yeast, principally Hanseniaspora and
Metschnikowia
– Non‐Saccharomyces yeasts present at levels of
105‐ 106 organisms/mL, Saccharomyces present at
102 ‐ 103 cells/mL
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Yeast and other microorganisms present
during fermentation of wine
• Fermentation depend on the presence of
naturally occurring yeast in must
t ll i ti t
• Use of SO2 treatment in must preparation
– Reduces total number of yeast and other
• S. cerevisiae is high SO2 resistance
• Moulds die quickly during fermentation
• Lactic acid bacteria are unable to compete for
Lactic acid bacteria are unable to compete for
fermentable sugar, but increase during
malolactic fermentation
– Decarboxylation of malic to lactic acid
– Deacidify the wine
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Inoculated fermentation
• Use of starter (S. cerevisiae) is common practice
in non‐traditional wine making
– Inoculated Fermentations
I l t dF t ti
• With active dry yeast (ADY)
• With a starter culture in juice
• With an already fermenting must/juice
• Level of Inoculum
– Typically 105 ‐ 106 cells/ml or 1 to 0.1% on a
volume/volume basis
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Inoculated vs Natural fermentation of
wine
Innoculated Natural
• The benefit: • The Benefits
– Predictability; Control of – Increased microbial
spoilage characteristics; complexity; Slower
Neutralityenhanced fermentation rates
varietal characteristics • The Negatives
• The Negatives – Off‐character formation; Lack
– Reduce
Reduce overall complexity;
overall complexity; of predictability; Seasonal
of predictability; Seasonal
Fermentation rates too rapid; variation in microbial
Wine too "yeasty“ (lack of populations on fruit
flavour)
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Sake
• Traditional Japanese drink
• Two stages of fermentation
Two stages of fermentation
– Koji
• Fermentation of glutinous rice by Aspergillus
oryzae
– Sake
• Fermented by Sacch. Cerevisiae which tolerant
to high concentration of etanol acid and sugar
to high concentration of etanol, acid and sugar
• Yeast starter culture: moto, contains lactic acid
bacteria, and can initiate fermentation at low T
(<10 C)
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Beer
• The oldest form of alcohol created by man.
• The ancient Mesopotamians and Sumerians were brewing as
early as 10,000 BC
• Clay tablets from 6,000 B.C. in Babylonia depict the beer
making process:
– The recipe utilized underbaked bread made from germinated barley.
– Being underbaked, the bread serves as a live yeast culture and when
the bread was cut into small pieces and placed in a large jug with
water malt would be produced
water, malt would be produced.
– The preinoculated malt when left out will ferment becoming beer.
• Beer production was considered women’s work.
• Refining of the beer process has developed over 5,000 ‐ 8,000
years
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Beers
• Yeast cannot ferment starch, only sugars, so
first starch must be converted to sugar
first starch must be converted to sugar.
• In malting germination of grain breaks
starch into maltose.
– barley is traditional grain used in
brewing
• Any grain, potatoes, or cassava can be used
Any grain potatoes or cassava can be used
as carbohydrate source.
• Beers average about 5% alcohol.
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Desirable properties of Brewer’s Yeast
• Bottom fermentation process (ale)
– S.cerevisiae
• Top fermentation process (lager)
– S. uvarum
• Carbohydrate utilization
– Increase metabolism of maltose and
Increase metabolism of maltose and
maltotriose in malt wort
– Ferment dextrin and starch
• Efficiency and low calorie (light) beer
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Five Main Ingredients
• Water
– Major ingredient, 95% of the content of beer
Major ingredient 95% of the content of beer
• Yeast
– Two main functions,
• Determines a beers flavour
• Responsible for converting sugar into alcohol
and carbon dioxide
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Five Main Ingredients
• Adjuncts
– Include corn or rice
– Added to produce a lighter tasting beer
• Malt
– Derived through barley malting process
– Imparts a rich mellow flavour in beer
• Hops
– Gives beer its fragrance, flavour and bitterness
• Mixture of beer ingredient is called wort
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Beer fermentation
• Top fermentation
– Used in production of ales
– CO2 carries flocculated Sacch. cerevisiae to the surface of the
fermenting vessel
– Produced at higher temperatures, 15 to 25 °C.
• gives for faster fermentation and beers with more fruity, aromatic
characteristics.
– The quantity of yeast used is lesser than for bottom
fermentation.
• Bottom fermentation.
B tt f t ti
– Used in production of lagers
– Yeast is injected at lower temperatures (6.5 to 8.5 °C)
– The yeast settles down
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Bread fermentation
Bread the earliest processed food
Manufactured from 3000 BC in Egypt
Weigh and mix Dough Dough

ingredient Fermentation
Bake Fermentation Portioned and

shaped
h d
Cool Sliced and packed
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Yeast Culture
• S. cerevisiae or baker yeast
• Properties and characteristics for bread making:
– Gassing power
– Flavor development
– Stable to drying
– Stable during storage
– Easy to dispense
– Ethanol tolerant
– Cryotolerant
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Microbiology of bread making
• Conventional bread making:
– S. Cerevisiae
• Bacteria:
– Commercial baker’s yeast about 5% contaminating LAB
• End Products:
– CO2
– Other compounds:
• Various organic acids and flavour compounds
• Factor affecting growth:
F t ff ti th
– T: 25‐28 C to minimize bacterial contaminnation
– RH: 70‐80%
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Q and A
• Microorganism responsible to produce ethanol from rice:
A. Saccharomyces cereviciae
B. Amylomyces rouxii
C. Penicillium
D. A and B correct
E. B and C correct
• During bread fermentation, the expected product is:
A. CO2
B
B. Alcohol
C. Biomass
D. A and C correct
E. B and C correct
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Thank you
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FerTech Lect 8 Alcohol

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FerTech Lect 8 Alcohol

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Tekfer14_Lecture8 9/3/2015

ITP 321 Lecture Note 8

In yeast, when O2 is available, cells use aerobic cellular

Acetaldehyde is an important component in many

Verstrepen et al. 2006

Preparation of tape starter culture: home scale

Inoculation with commercial tape starter

The Basic Steps of Red Wine

The Basic Steps of White Wine

Grape is just a right substrat

Weigh and mix Dough Dough

Bake Fermentation Portioned and

Cool Sliced and packed

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